1. Field of the Invention
The present invention relates to novel proteins involved in the initiation of eukaryotic transcription. More specifically, isolated nucleic acid molecules are provided encoding a human Prt1-like subunit protein (hPrt1) and a human eIF4G-like protein (p97). Also provided are hPrt1 and p97 polypeptides, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of hPrt1 and p97 activity.
2. Related Art
Eukaryotic protein synthesis requires the participation of translation initiation factors, which assist in the binding of the mRNA to the 40S ribosomal subunit (reviewed in Merrick & Hershey, in Translational Control, Hershey et al., eds., Cold Spring Harbour Laboratory Press, (1996), pp. 31-69 and Pain, Eur. J. Biochem 236:747-771 (1996)). Ribosome binding is facilitated by the cap structure (m.sup.7 GpppN, where N is any nucleotide) that is present at the 5' end of all cellular mRNAs (except organellar). Biochemical fractionation studies elucidated the general pathway for translation initiation and led to the characterization of several translation initiation factors (reviewed in Merrick & Hershey supra). It is believed that the mRNA cap structure is initially bound by eukaryotic initiation factor (eIf) 4F, which, in conjunction with eIF4B, melts RNA secondary structure in the 5' untranslated region (UTR) of the mRNA to promote ribosome binding. eIF4F is a more efficient RNA helicase than free eIF4A (Rozen et al., Mol. Cell. Biol. 10:1134-1144 (1990)), consistent with the idea that eIF4A recycles through the eIF4F protein complex to function in unwinding (Pause et al., Nature 371:762-767 (1994)). The 40S ribosomal subunit, in a complex with eIF3, eIF1A and eIF2-GTP-tRNAimet, binds at or near the cap structure and scans vectorially the 5' UTR in search of the initiator AUG codon (reviewed in Merrick & Hershey, supra).
eIF3 is the largest translation initiation factor, with at least 8 different polypeptide subunits and a total mass of approximately 550 to 700 kDa (Schreier, et al., J. Mol. Biol. 116:727-753 (1977); Benne & Hershey, Proc. Natl. Acad. Sci. USA 73:3005-3009 (1976); Behlke et al., Eur. J. Biochem. 157:523-530 (1986)). In mammals, the apparent molecular masses of the eIF3 subunits are 35, 36, 40, 44, 47, 66, 115 and 170 kDa (Behlke, supra; Meyer, et al., Biochemistry 21:4206-4212 (1982); Milburn et al., Arch. Biochem. Biophys 276:6-11 (1990)). eIF3 is a moderately abundant translation initiation factor, with 0.5 to 1 molecule per ribosome in HeLa cells and rabbit reticulocyte lysates (Meyer, supra; Mengod & Trachsel, Biochem. Acta 825:169-174 (1985)). This protein complex assumes several functions during translation initiation (reviewed in Hannig, BioEssays 17:915-919 (1995)). eIF3 binds to the 40S ribosomal subunit and prevents joining with the 60S subunit. It interacts with the ternary complex and stabilizes the binding of the latter to the 40S ribosomal subunit (Trachsel et al., J. Mol. Biol. 116:755-767 (1977); Gupta et al., (1990); Goumans et al., Biochem. Biophys. Acta 608:39-46 (1980); Peterson et al., J. Biol. Chem. 254:2509-2510 (1979)). eIF3 crosslinks to mRNA and 18S rRNA (Nygard & Westermann, Nucl. Acids Res. 10:1327-1334 (1982); Westermann & Nygard, Nucl. Acids Res. 12:8887-8897 (1984)), an activity mainly attributed to the 66 kDa subunit (or 62 kDa in yeast; Garcia-Barrio, et al., Genes Dev. 9:1781-1796 (1995); Naranda, et al., J. Biol. Chem. 269:32286-32292 (1994)). eIF3 co-purifies with eIF4F and eIF4B, two initiation factors involved in the mRNA binding step (Schreier et al., J. Mol. Biol. 116:727-753 (1977)). A direct interaction between the 220 kDa subunit of eIF4F and eIF3 has been demonstrated (Lamphear et al., J. Biol. Chem. 270:21975-21983 (1995)) and a role for eIF3 serving as a bridge between the 40S ribosomal subunit and eIF4F-bound mRNA has been postulated (Lamphear, supra).
The complex structure of eIF3 and its pleiotropic roles in translation initiation have rendered the study of this factor difficult. The protein sequence for only three of the yeast subunits (SUI1/p16, p62 and PRT1/p90) have been published (Garcia-Barrio et al., Genes Dev. 9:1781-1796 (1995); Naranda, supra; Hanic-Joyce et al., J. Biol. Chem. 262:2845-2851 (1987)). However, several other mammalian and yeast subunits have been recently cloned. The yeast protein p90, also known as Prt1, is the most well characterized of those identified to date. Prt1 is an integral subunit of eIF3 (Naranda, supra; Danaie et al., J. Biol. Chem. 270:4288-4292 (1995)). A conditional lethal mutation in the PRT1 gene reduces the binding of the ternary to the 40S ribosomal subunit (Feinberg et al., J. Biol. Chem. 257:10846-10851 (1982)). Other mutations which confer temperature sensitivity are located in the central and carboxy-terminal portion of Prt1. An N-terminal deletion which removes the Prt1 putative RNA Recognition Motif (RRM; for reviews see Bimey, et al., Nucl. Acids Res. 21:5803-5816 (1993); Burd & Dreyfuss, Science 265:615-621 (1994b); Nagai et al., Trends Biochem. Sci. 20:235-240 (1995)), acts a trans-dominant negative inhibitor (Evans et al., Mol. Cell. Biol. 15:4525-4535 (1995)).
Proteins that specifically inhibit cap-dependent translation have been described (Pause, supra; Lin et al., Science 266:653-656 (1994)): 4E-binding protein-1 and -2 (4E-BP1 and 4E-BP2) bind to eIF4E and prevent their association with eIF4G, because 4E-BPs and eIF4G share a common site for eIF4E binding (Haghighat et al., EMBO J. 14:5701-5709 (1995); Mader et al., Mol. Cell. Biol. 15:4990-4997 (1995)). Upon treatment of cells with insulin and growth factors, 4E-BPs become phosphorylated. This leads to dissociation of the 4E-BPs from eIF4E and formation of the eIF4F complex, which results in stimulation of translation (Pause, supra; Lin, supra; Beretta, et al., EMBO J. 15:658-664 (1996)).